A Novel Anti-CD44 Variant 3 Monoclonal Antibody C44Mab-6 Was Established for Multiple Applications

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3–10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3–10, COLO205, and HSC-3 was 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M, respectively. C44Mab-6 could detect the CD44v3–10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.


Introduction
The cell surface glycoprotein known as cluster of differentiation 44 (CD44) is broadly expressed by epithelial, mesenchymal, and hematopoietic cells and is involved in adhesion to the extracellular matrix (ECM), lymphocyte homing, and lymphocyte activation [1]. A growing body of evidence reveals the critical roles of CD44 in tumor progression and metastasis [2,3]. The human CD44 gene consists of 19 exons, 10 of which are constant in all variants, and makes up the standard form of CD44 (CD44s) [4]. Furthermore, a large number of CD44 variants (CD44v) are generated due to alternative splicing. The CD44v consists of 10 constant exons in combination with the remaining 9 variant exons.

The Reactivity of C 44 Mab-6 to CD44-Expressing Cells in Flow Cytometry
The reactivity of C 44 Mab-6 to CHO/CD44v3-10, CHO/CD44s, and CHO-K1 cells was investigated by using flow cytometry. C 44 Mab-6 dose-dependently recognized CHO/ CD44v3-10 cells ( Figure 3A). In contrast, C 44 Mab-6 recognized neither CHO/CD44s ( Figure 3B) nor CHO-K1 ( Figure 3C) cells. C 44 Mab-46, which is an anti-pan-CD44 mAb [30], recognized both CHO/CD44v3-10 and CHO/CD44s cells (Supplementary Figure S1). We next examined the reactivity of C 44 Mab-6 to a colorectal cancer cell line (COLO205) and an OSCC cell line (HSC-3). COLO205 was selected in this study from various cancer cell lines because C 44 Mab-6 showed very high reactivity to it. Furthermore, HSC-3 was selected because HNSCC was shown to be the second highest CD44-expressing cancer type in the Pan-Cancer Atlas [39]. C 44 Mab-6 could recognize a colorectal cancer cell line COLO205 ( Figure 3D) and an oral squamous cell line HSC-3 ( Figure 3E) in a dose-dependent manner.

Immunohistochemical Analysis by Using C44Mab-6 against Tumor Tissues
Immunohistochemical analysis against the formalin-fixed paraffin-embedded (FFPE) sections of OSCC was conducted to assess the availability of C44Mab-6. We used sequential sections of OSCC tissue microarray and compared the staining patterns of C44Mab-6 and C44Mab-46. Clear membranous staining was observed for C44Mab-6 and C44Mab-46 in a well-differentiated OSCC section ( Figure 6A,B). Figure 6C,D showed an OSCC section with the stromal invaded phenotype. C44Mab-6 strongly stained stromal-invaded OSCC and could clearly distinguish tumor cells from the surrounding stroma cells ( Figure 6C). In contrast, C44Mab-46 stained both invaded tumor and stromal cells ( Figure 6D). In Figure  6E and F, C44Mab-6 partially stained tumor cells but not stromal cells ( Figure 6E). In contrast, C44Mab-46 mainly stained stromal cells ( Figure 6F). We summarized the data of immunohistochemical analysis of CD44 expression in tumor cells in Table 1; C44Mab-6 stained 44 out of 50 (88%) cases of OSCC. We also stained FFPE sections of colorectal cancer tissue microarray and found that C44Mab-6 stained 7 out of 40 (18%) cases (Supplementary Table S2). However, the C44Mab-6 reactivity was faint and partly localized compared to that of C44Mab-46 (Supplementary Figure S2). These results indicated that C44Mab-6 is useful for the immunohistochemical analysis of FFPE tumor sections.

Immunohistochemical Analysis by Using C 44 Mab-6 against Tumor Tissues
Immunohistochemical analysis against the formalin-fixed paraffin-embedded (FFPE) sections of OSCC was conducted to assess the availability of C 44 Mab-6. We used sequential sections of OSCC tissue microarray and compared the staining patterns of C 44 Mab-6 and C 44 Mab-46. Clear membranous staining was observed for C 44 Mab-6 and C 44 Mab-46 in a well-differentiated OSCC section ( Figure 6A,B). Figure 6C,D showed an OSCC section with the stromal invaded phenotype. C 44 Mab-6 strongly stained stromal-invaded OSCC and could clearly distinguish tumor cells from the surrounding stroma cells ( Figure 6C). In contrast, C 44 Mab-46 stained both invaded tumor and stromal cells ( Figure 6D). In Figure 6E,F, C 44 Mab-6 partially stained tumor cells but not stromal cells ( Figure 6E). In contrast, C 44 Mab-46 mainly stained stromal cells ( Figure 6F). We summarized the data of immunohistochemical analysis of CD44 expression in tumor cells in Table 1; C 44 Mab-6 stained 44 out of 50 (88%) cases of OSCC. We also stained FFPE sections of colorectal cancer tissue microarray and found that C 44 Mab-6 stained 7 out of 40 (18%) cases (Supplementary Table S2). However, the C 44 Mab-6 reactivity was faint and partly localized compared to that of C 44 Mab-46 (Supplementary Figure S2). These results indicated that C 44 Mab-6 is useful for the immunohistochemical analysis of FFPE tumor sections. Figure 6. Immunohistochemical analysis by using C44Mab-6 and C44Mab-46 against OSCC tissues. After antigen retrieval, serial sections of OSCC tissue array (Catalog number: OR601c) were incubated with 1 μg/mL of C 44 Mab-6 (A,C,E) or 1 μg/mL of C 44 Mab-46 (B,D,F), followed by treatment with the Envision+ kit. The chromogenic reaction was conducted by using 3,3′-diaminobenzidine tetrahydrochloride (DAB). The counterstaining was performed by using hematoxylin. Scale bar = 100 μm.

Discussion
In this study, we developed C 44 Mab-6 by using the CBIS method ( Figure 1) and determined its epitope as a variant 3-encoded region of CD44 (Figure 2 and Supplemental Table S1). Then, we showed the usefulness of C 44 Mab-6 for multiple applications, including flow cytometry (Figures 3 and 4), Western blotting ( Figure 5), and immunohistochemistry ( Figure 6).
An anti-CD44v3 mAb (clone 3G5) was previously developed and widely used for various applications [40]. The 3G5 was developed by the immunization of COS1-produced CD44v3-10-Fc protein. The specificity to the exon was determined via indirect immunofluorescent staining of COS1 cells which expressed CD44v3-10, CD44v6-10, CD44v7-10, CD44v8-10, and CD44v10 [40]. Therefore, the 3G5 is thought to recognize the peptide or glycopeptide structure of CD44v3. However, the detailed binding epitope of 3G5 has not been determined. As shown in Supplementary Table S1, C 44 Mab-6 recognized CD44 p231-250 peptide (AGWEPNEENEDERDRHLSFS) but not CD44 p241-260 peptides (DERDRHLSFSGSGIDDDEDF). The underlined SGSG sequence is a heparan sulfatemodified sequence in the variant-3-encoded region [7,12]. Therefore, the recognition of C 44 Mab-6 is probably not affected by the heparan sulfate modification.
Head and neck cancers are derived mainly from the oral cavity, larynx, pharynx, and nasal cavity [41]. SCC is the common type. As shown in Figure 6, C 44 Mab-6 clearly stained the membrane of OSCC and recognized a human OSCC cell line, namely, HSC-3 (Figures 3 and 4). The CD44v3-high and aldehyde dehydrogenase-1 (ALDH1)-high population of HSC-3 exhibited a potent tumorigenic potential in immunodeficient NOD/SCID mice [42]. The population showed increased stemness-related transcriptional factors, including OCT4, SOX2, and NANOG [42]. In a future study, we will investigate the application of C 44 Mab-6 to isolate cancer stem-like cells from cancer cell lines and/or OSCC tissues. We will further establish the strategy to deplete the cancer stem-like cells for tumor therapy. We have just started the cDNA cloning of C 44 Mab-6 heavy and light chains for therapeutic application. We have investigated the antitumor activity by using class-switched and defucosylated IgG 2a mAbs [34,[43][44][45][46][47][48][49]. The defucosylated IgG 2a mAbs can be produced by fucosyltransferases 8-deficient CHO-K1 cells, exhibited potent ADCC activity in vitro, and suppressed the xenograft growth [34,[43][44][45][46][47][48][49]. Therefore, the production of defucosylated C 44 Mab-6 is one of the strategies to evaluate antitumor activity in vivo.
The HNSCC treatments include surgery, chemotherapy, radiotherapy, molecular targeted therapy, immunotherapy, or a combination of these modalities [50]. Despite the progress of the therapies, drug resistance and metastasis are still the main causes of death [51]. In a preclinical study, a pan-CD44 mAb was applied to the novel modalities, including near-infrared photoimmunotherapy (NIR-PIT). The CD44 mAb-photoactivatable dye IRDye700DX conjugate exhibited significant antitumor effects after the NIR-light exposure against CD44-expressing OSCC [52]. However, a pan-CD44 mAb, namely, C 44 Mab-46, recognized not only tumor cells but also stromal tissues ( Figure 6D,F) and probably immune cells, which are important for antitumor immunity. Therefore, CD44v is a more rational tumor antigen for NIR-PIT, which could be a new modality for OSCC with locoregional recurrence.
Zen et al. established a unique mAb (clone C3H7), which recognized the basolateral membranes of epithelium and inhibited both the adhesion of epithelial cells to immobilized CD11b/CD18 and the transepithelial migration of leukocytes [53]. CD11b/CD18, also known as Macrophage-1 antigen, is a leukocyte integrin that is essential for firm adhesion to epithelial cells and the transepithelial migration of leukocytes [54]. However, the receptor of CD11b/CD18 on epithelial cells has not been identified. They revealed that the antigen of C3H7 is CD44v3, which specifically binds to CD11b/CD18 through its heparan sulfate moieties [53]. The C3H7 antigen was increased via treatment with pro-inflammatory cytokine, including interferon-γ and tumor necrosis factor-α in epithelial monolayers [53], which supports the previous finding that CD44v3 is increased in inflammatory diseases, including ulcerative colitis [55]. C 44 Mab-6 also recognized the basolateral surface of colorectal cancer cells (Supplementary Figure S2A). Further investigations are required for the relationship between CD44v3 expression and the transepithelial migration of leukocytes. The study could provide the basis for the development of novel therapeutic applications of anti-CD44v3 mAbs, including C 44 Mab-6.
Chimeric antigen receptor T-cell (CAR-T) therapies have been developed for a variety of hematopoietic malignances and solid tumors [56]. CAR-T cells have demonstrated remarkable success in treating CD19 + B cell leukemias [57]. However, CAR-T therapy for acute myeloid leukemia (AML) has been elusive because of target restriction and phenotypic heterogeneity [58]. Mutations of the FMS-like tyrosine kinase 3 (FLT3) and DNA methyltransferase 3A (DNMT3A) genes were identified as common driver mutations associated with poor prognosis of AML patients [59]. Tang et al. showed that AML cells expressed high levels of CD44 mRNA, and the expression of AML-derived FLT3 and DNMT3A mutants promote the transcription of CD44 mRNA through suppression of CpG island methylation in the CD44 promoter [60]. They also found that AML patients with FLT3 or DNMT3A mutations had higher expression of CD44v6 compared to normal specimens. Furthermore, they showed that CD44v6 CAR-T cells exhibited potent antileukemic effects [60]. Therefore, CD44v6 is thought to be a rational antigen of CAR-T therapy for AML with FLT3 or DNMT3A mutations.
Since CD44 mRNA is upregulated in AML, there is a possibility that other CD44 variants are also transcribed and expressed in AML. In a humanized mouse model of chronic myeloid leukemia (CML) progression from chronic phase to blast crisis, a CD44 variant (CD44v8-10) was elevated, which is required for the maintenance of stemness [61]. Although we have examined the reactivity of C 44 Mab-6 against cell lines derived from hematopoietic malignancy and found increased reactivity in several cell lines, further studies are required for the selective expression of CD44v3 in leukemia cells, but not in hematopoietic stem cells, to ensure its safety as a CAR-T antigen.

Production of Hybridomas
All animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001). The female BALB/c mice (CLEA Japan, Tokyo, Japan) were intraperitoneally immunized with CHO/CD44v3-10 (1 × 10 8 cells) with Imject Alum (Thermo Fisher Scientific Inc.) as an adjuvant. The three additional immunizations per week and a booster injection were performed two days before the harvest of the spleen cells. The hybridomas were produced via the fusion of splenocytes and P3U1 cells by using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA). The supernatants, which were positive for CHO/CD44v3-10 cells and negative for CHO-K1 cells, were selected by using the SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).

Determination of Dissociation Constant (K D ) via Flow Cytometry
In CHO/CD44v3-10 cells, we prepared from 130 to 0.008 nM (diluted by 1/2) of C 44 Mab-6. In COLO201 and HSC-3 cells, we prepared from 1300 to 0.08 nM (diluted by 1/2) of C 44 Mab-6. The serially diluted C 44 Mab-6 was suspended with 2 × 10 5 cells. Then, the cells were incubated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:200). BD FACSLyric and BD FACSuite software version 1.3 (BD Biosciences, Franklin Lakes, NJ, USA) were used for the fluorescence data analyses. The K D was determined by the fitting binding isotherms to built-in one-site binding models of GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA).

Immunohistochemical Analysis
FFPE sections of OSCC tissue array (OR601c) and colorectal carcinoma tissue array (CO483a) were purchased from US Biomax Inc. (Rockville, MD, USA). The tissue arrays were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C 44 Mab-6 (1 µg/mL) and C 44 Mab-46 (1 µg/mL) for 1 h at room temperature. The sections were further incubated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min. Then, a chromogenic reaction using 3,3 -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) was conducted. Hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used for the counterstaining. To examine the sections and obtain images, we used Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).